microM Min Research Articles

Progressive inactivation of plasma membrane (Ca2++Mg2+)ATPase by Cd2+ in the absence of ATP and reversible inhibition during catalysis.

The influence of Cd2+ ions on plasma membrane (Ca2++Mg2+)ATPase activity from red cells was investigated. When the membranes were preincubated with Cd2+ in the absence of ATP, basal (Ca2++Mg2+)ATPase activity (no calmodulin) was slowly and irreversibly inhibited (inactivated) following first-order kinetics (k/Ki = 0.0057 microM-1 min-1 at [Cd2+] = 0.25-1 microM). However, preincubation with Cd2+ did not affect the degree of stimulation by calmodulin added to the assay medium together with ATP. Inactivation was not released by prolonged exposure of membranes to EGTA prior to catalysis, but it was strongly attenuated when the pH in the preincubation medium was lowered from 7.2 to 6.4. When the reaction was started by supplying membranes simultaneously with Cd2+ and ATP (no preincubation), (Ca2++Mg2+)ATPase was inhibited by increasing concentrations of the CdATP complex ([CdATP]50 = 7.2 microM). In this condition, however, even total inhibition of the pump was almost completely released after addition of enough EGTA to decrease CdATP concentrations to the nanomolar range. These results, taken together, indicate that inactivation of the unphosphorylated enzyme by Cd2+ is influenced by dissociation of amino acid residues exhibiting pK between 6.0 and 7.0, and that recognition by the pump of the physiological modulator calmodulin is preserved in the preincubated pump molecules which did not undergo inactivation. In addition, they show that the catalytic site is a target for reversible inhibition of the pump by CdATP and that occupancy of the nucleotide binding site prevents inactivation.

Use of time-lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis.

We describe the use of time-lapse fluorescence microscopy to visualize the movement of the DNA replication origin and terminus regions on the Bacillus subtilis chromosome during the course of the cell cycle. The origin and terminus regions were tagged with a cassette of tandem lac operator repeats and visualized through the use of a fusion of the green fluorescent protein to the LacI repressor. We have discovered that origin regions abruptly move apart towards the cell poles during a brief interval of the cell cycle. This movement was also seen in the absence of cell wall growth and in the absence of the product of the parB homologue spo0J. The origin regions moved apart an average distance of 1.4 microm in an 11 min period of abrupt movement, representing an average velocity of 0.17 microm min(-1), and reaching a maximum velocity of greater than 0.27 microm min(-1). The terminus region also exhibited a striking pattern of movement but not as far or a rapid as the origin region. These results provide evidence for a mitotic-like motor that is responsible for segregation of the origin regions of the chromosomes.

Interaction of Rac1 with GTPase-activating Proteins and Putative Effectors: A COMPARISON WITH Cdc42 AND RhoA

The intrinsic GTPase activity of the Rho family GTP-binding protein Rac1 is drastically stimulated upon interaction with its GTPase-activating proteins (GAPs) and is significantly inhibited when coupled to certain effector targets such as the p21-activated kinases (PAKs) and IQGAPs. Here we have characterized the interaction of Rac1 with a panel of mammalian GAPs and putative effectors by measuring the kinetic and binding parameters involved and made comparisons with similar interactions for Cdc42 and RhoA. In contrast with Cdc42 (for which the GAP domain of p50RhoGAP is 50-fold more efficient than those of p190, Bcr, and 3BP-1) and with RhoA (toward which only p50RhoGAP and p190 displayed high efficiencies), the catalytic efficiencies (Kcat/Km) of the GAP domains of p50RhoGAP, p190, Bcr, and 3BP-1 on Rac1 are found to be comparable in a range between 0.9 and 2.6 min-1 microM-1. However, similar to the cases of Cdc42 and RhoA, the Km values of the GAP domains on Rac1 compare well to the binding affinity to the guanylyl imidodiphosphate-bound Rac1, which ranges from 10.5 to 40.5 microM, suggesting a rapid equilibrium reaction mechanism. The dissociation constants of the p21-binding domains of PAK1, PAK2, and the RasGAP-related domain of IQGAP1, which all cause significant reduction of the intrinsic rate of GTP hydrolysis upon binding to Rac1-GTP, are found to be 0.71, 0.26, and 2.13 microM for Rac1-GTP, compared with that determined for Cdc42-GTP at 2.9, 20.5, and 0.39 microM, respectively, under similar conditions. These results suggest that p50RhoGAP, p190, Bcr, and 3BP-1 are all capable of acting as a negative regulator for Rac1-mediated signaling, and that, although PAK1 and IQGAP1 can couple tightly with both Rac1 and Cdc42, PAK2 is likely to be a specific effector for Rac1 instead of Cdc42.

Vitamin K2 (menaquinone) biosynthesis in Escherichia coli: evidence for the presence of an essential histidine residue in o-succinylbenzoyl coenzyme A synthetase.

o-Succinylbenzoyl coenzyme A (OSB-CoA) synthetase, when treated with diethylpyrocarbonate (DEP), showed a time-dependent loss of enzyme activity. The inactivation follows pseudo-first-order kinetics with a second-order rate constant of 9.2 x 10(-4) +/- 1.4 x 10(-4) microM(-1) min(-1). The difference spectrum of the modified enzyme versus the native enzyme showed an increase in A242 that is characteristic of N-carbethoxyhistidine and was reversed by treatment with hydroxylamine. Inactivation due to nonspecific secondary structural changes in the protein and modification of tyrosine, lysine, or cysteine residues was ruled out. Kinetics of enzyme inactivation and the stoichiometry of histidine modification indicate that of the eight histidine residues modified per subunit of the enzyme, a single residue is responsible for the enzyme activity. A plot of the log reciprocal of the half-time of inactivation against the log DEP concentration further suggests that one histidine residue is involved in the catalysis. Further, the enzyme was partially protected from inactivation by either o-succinylbenzoic acid (OSB), ATP, or ATP plus Mg2+ while inactivation was completely prevented by the presence of the combination of OSB, ATP, and Mg2+. Thus, it appears that a histidine residue located at or near the active site of the enzyme is essential for activity. When His341 present in the previously identified ATP binding motif was mutated to Ala, the enzyme lost 65% of its activity and the Km for ATP increased 5.4-fold. Thus, His341 of OSB-CoA synthetase plays an important role in catalysis since it is probably involved in the binding of ATP to the enzyme.

Nuclear traffic in fungal hyphae: in vivo study of nuclear migration and positioning in Aspergillus nidulans.

Nuclear migration and nuclear positioning are fundamental processes in all eukaryotic cells. They are easily monitored during hyphal growth of filamentous fungi. We expressed the green fluorescent protein (GFP) as a fusion protein with the putative nuclear localization domain of the transcriptional activator stuA in nuclei of Aspergillus nidulans and visualized these organelles in living cells. Nuclear staining was observed in interphase nuclei but not during mitosis. Nuclear division, nuclear migration, septum formation and branching were analysed with time-lapse video microscopy during hyphal extension. Hyphae elongated at 0.1-1.2 microm min(-1) and nuclei moved with similar speeds towards the hyphal tip until they had reached a defined position. An individual regulation of nuclear mobility in a given hyphal compartment was observed. Some representative movies are available on the Internet (http://www.blacksci.co.uk/products/journals/molextra.htm). Nuclear positioning was also studied at the molecular level. The ApsA protein, which regulates nuclear migration, was localized at the cytoplasmic membrane in germlings and hyphae by immunofluorescence and GFP tagging. A model of nuclear migration, nuclear positioning and the role of ApsA is presented.

Isolation of hammerhead ribozymes with altered core sequences by in vitro selection.

The hammerhead ribozyme has an invariant nucleotide sequence in the core region. In order to search for alternative sequences which can support the cleavage after the triplet GUC, the core region of 10 nucleotides was randomized and subjected to in vitro selection by repeated cycles of transcription, reverse transcription, and PCR. Active sequences were isolated after each transcription by denaturing PAGE, and after nine cycles of selection, two sequences dominated the pool. Both sequences conformed broadly to the consensus core region except that in one sequence a single A9U mutation was observed while in the other two mutations at A9U and U7A were seen. The catalytic efficiencies of these ribozymes were 6.4 and 14.1 microM(-1) min(-1), respectively, as compared to 163 microM(-1) min(-1) for the consensus sequence. Interestingly, the consensus was not found in any of the selected sequences. This discrimination against the consensus sequence was attributed to the specificity of the enzymes used in the selection procedure.

Crosslinking kinetics of the human transglutaminase, factor XIII[A2], acting on fibrin gels and gamma-chain peptides.

Factor XIII is the terminal enzyme of the coagulation cascade which serves to rapidly crosslink the adjacent gamma-chain C-termini of fibrin clots. In vivo, this process is initiated by the proteolytic action of thrombin which simultaneously converts both soluble fibrinogen to fibrin and activates zymogen FXIII; fibrin then spontaneously polymerizes to form a gel which activated FXIII stabilizes through crosslinking. Due to the kinetic complexity and the difficulty of investigating gel phase reactions, methods employing pre-activation of recombinant human Factor XIII (rFXIII[A'2]) were developed to effectively decouple these reactions. By utilizing these methods, the kinetic parameters of gamma-chain crosslinking in fibrin gels could be determined by both initial rate and integrated rate techniques under physiologically relevant conditions. The crosslinking of the gamma-chain of fibrin gels could be described by apparent Michaelis kinetics with K(m)(app) = 6.2 microM, kcat = 1872 min-1, and Ksp = 302 min-1 microM-1 for a fibrin gamma-chain monomer of M(r) = 170000 Da. In contrast, both the crosslinking rates of alpha-chains within fibrin gels (Ksp = 0.38 min-1 microM-1: Bishop et al. (1993)) and the crosslinking of a soluble synthetic peptide containing the unique gamma-chain fibrin crosslinking site (Ksp = 0.030 min-1 microM-1) could not be shown to saturate and gave apparent first-order rates with respect to rFXIII[A'2]. These observations coupled with the large differences in the turnover rates (approximately 10(4)) suggest two likely mechanisms for FXIII[A'2]-substrate interactions: (1) random (or independent) binding of non- or weakly interacting substrate pairs imposes a high entropic barrier (i. e., delta Gbinding) to the formation of a productive catalytic complex, e.g., for soluble gamma-chain peptides and the flexible alpha-chains within fibrin, and (2) binding to an oriented substrate pair effectively lowers the entropic barrier to formation of a Michaelis complex and thus greatly enhances the rate of catalysis, e.g., for gamma-chain pairs within the fibrin fibrils.

Aminoacyl and peptidyl analogs of chloramphenicol as slow-binding inhibitors of ribosomal peptidyltransferase: a new approach for evaluating their potency.

In a model system derived from Escherichia coli, acetylphenylalanyl-puromycin is produced in a pseudo-first-order reaction between the preformed acetylphenylalanyl/tRNA/poly(U)/ribosome complex (complex C) and excess puromycin. Two aminoacyl analogs [3, Gly-chloramphenicol (CAM): 4, L-Phe-CAM] and two peptidyl analogs (2, L-Phe-Gly-CAM: 5, Gly-Phe-CAM) of CAM (1) were tested as inhibitors in this reaction. Detailed kinetic analysis suggests that these analogs (I) react competitively with complex C and form the complex C*l, which is inactive toward puromycin. C*l is formed via a two-step mechanism in which C*l is the product of a slow conformational change of the initial encounter complex Cl according to the equation C + l reversible Cl reversible C*l. Furthermore, we provide evidence that analog 5 may react further with C*l forming the species C*l2. The values of the apparent association rate constant (K(assoc)) are 1.42 x microM-1 min-1 for 2, 0.55 x microM-1 min-1 for 3, and 0.18 x microM-1 min-1 for 4 and 0.038 x microM-1 min-1 for 5 [corrected]. In the case of analog 5, K(assoc) is a linear function of the inhibitor concentration; when [I] approaches zero, the K(assoc) value is equal to 3.8 x 10(2) M-1 sec-1. Such values allow the classification of CAM analogs as slow-binding inhibitors. According to K(assoc) values, we could surmise that analog 2 is 2.5-fold more potent than 3 and 8-fold more potent than 4. The relative potency of analog 5 is the lowest among the analogs and is dependent on its concentration. The results are compared with previous data and discussed on the basis of a possible retro-inverso relationship between CAM analogs and puromycin.

Catalytic Mechanism and DNA Substrate Recognition of Escherichia coli MutY Protein

Escherichia coli MutY protein cleaves A/G- or a/7,8-dihydro-8-oxo-guanine (A/GO)-containing DNA on the A-strand by N-glycosylase and apurinic/apyrimidinic endonuclease or lyase activities. In this paper, we show that MutY can be trapped in a stable covalent enzyme-DNA intermediate in the presence of sodium borohydride, a new finding that supports the grouping of MutY in that class of DNA glycosylases that possess concomitant apurinic/apyrimidinic lyase activity. To potentially help determine the substrate recognition site of MutY, mutant proteins were constructed. MutY proteins with a Gly116 --> Ala (G116A) or Asp (G116D) mutation had reduced binding affinities for both A/G- and A/GO-containing DNA substrates. The catalytic parameters, however, were differentially affected. While A/G- and A/GO-containing DNA were cleaved by MutY with specificity constants (kcat/Km) of 10 and 3.3 min-1 microM-1, respectively, MutY(G116D) cleaved these DNAs 2, 300- and 9-fold less efficiently. The catalytic activities of MutY(G116A) with A/G- and A/GO-containing DNA were about the same as that of wild-type MutY. Both MutY(G116A) and MutY(G116D) could be trapped in covalent intermediates with A/GO-containing DNA, but with lower efficiencies than the wild-type enzyme in the presence of sodium borohydride. MutY(G116A) also formed a covalent intermediate with A/G-containing DNA, but MutY(G116D) did not. Since Gly116 of MutY lies in a region that is highly conserved among several DNA glycosylases, it is likely this conserved region is in the proximity of the substrate binding and/or catalytic sites.

Substrate-based inhibitors of lanosterol 14.alpha.-methyl demethylase: II. Time-dependent enzyme inactivation by selected oxylanosterol analogs

Selected 15-, 32-, and 15,32-substituted lanosterol analogs are shown here to display time-dependent inactivation and lanosterol 14 alpha-methyl demethylase. These molecules are competitive with respect to substrate and require NADPH and O2 in order to display time dependence, thus supporting the premise that they are mechanism-based inactivators. Structural features required for lanosterol demethylation by the lanosterol demethylase such as nuclear double bond location and availability of an abstractable 15 alpha-proton are also essential elements for time-dependent inactivation. 32-(S)-Vinyllanost-8-en-3 beta,32-diol is a potent time-dependent inactivator (Kinact/Ki = 0.36 min-1 microM-1), while the 32-(R)-vinyllanost-8-en-3 beta,32-diol functions solely as a competitive demethylase inhibitor. These results support the premise that stereoselective oxidation occurs during lanosterol demethylation and that the 32-pro-S proton is abstracted during the demethylation reaction.

Extracellular Conversion of Epidermal Growth Factor (EGF) to des-Arg53-EGF by Carboxypeptidase M

Epidermal growth factor (EGF) is a 53-amino-acid mitogenic polypeptide present in a variety of tissues and fluids including kidney, urine, and amniotic fluid. An EGF isoform, des-Arg53-EGF, has been identified in urine and is the earliest metabolite generated in target cells upon EGF binding. In this study, purified carboxypeptidase M efficiently released the COOH-terminal arginine residue from EGF with a Km = 56 microM, kcat = 388 min-1, and kcat/Km = 6.9 microM-1 min-1. When EGF was incubated with urine or amniotic fluid, des-Arg53-EGF was the only metabolite detected. This conversion was blocked by immunoprecipitation with specific antiserum to carboxypeptidase M or by 10 microM DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (a carboxypeptidase M inhibitor), indicating that the major EGF metabolizing enzyme in these fluids is carboxypeptidase M. When incubated on a confluent monolayer of Madin-Darby canine kidney (MDCK) cells, EGF was readily converted to a single metabolite, des-Arg53-EGF, by carboxypeptidase M. To investigate one possible functional consequence of this conversion, mitogenic activities of EGF and des-Arg53-EGF were tested. Both peptides were equipotent in stimulating [3H]thymidine incorporation in MDCK cells at all doses tested. In addition, inhibition of the conversion of EGF to des-Arg53-EGF by the carboxypeptidase M inhibitor did not affect the mitogenic potency of EGF. These data indicate that carboxypeptidase M, present in a variety of cells and biological fluids, can convert EGF to des-Arg53-EGF. However, in contrast to many other peptide hormones whose activity depends on a final carboxypeptidase processing step, removal of Arg53 of EGF is not required for its mitogenic activity.

Renaturation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides after denaturation in 4 M guanidine hydrochloride: kinetics of aggregation and reactivation.

In 4 M guanidine hydrochloride (GdnHCl), the dimeric enzyme glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (G6PD) dissociated to subunits and was extensively unfolded. Rapid dilution of this high GdnHCl concentration allowed G6PD to partially renature, as measured by enzyme reactivation, to a level which depended on the conditions employed. The fraction of the enzyme which did not renature aggregated and precipitated out of solution, a process which could not be substantially prevented by stabilizing additives. Based on the enzyme concentration dependence of the reactivation yield and on a comparison of the aggregation and reactivation rates, it was determined that aggregation and reactivation compete kinetically for a partially-folded intermediate only very early in the process, during the rapid GdnHCl-dilution step. The kinetics of G6PD reactivation were sigmoidal, indicating that this process involves more than one rate-limiting reaction. The kinetics depended on enzyme concentration in a higher than first-order manner, indicating that association of subunits is one of the rate-limiting reactions. A renaturation mechanism compatible with these observations is described, which involves a bi-unimolecular (subunit association-folding) reaction sequence, with rate constants equal to 2.19 microM-1 min-1 and 0.140 min-1, respectively. This mechanism involves an inactive, dimeric, G6PD-folding intermediate, a species whose existence has recently been established by equilibrium denaturation experiments (Plomer, J.J. and Gafni, A. (1992) Biochim. Biophys. Acta 1122, 234-242).

Replication of the base pair 6-thioguanine/5-methyl-2-pyrimidine with the large Klenow fragment of Escherichia coli DNA polymerase I.

The kinetics and the fidelity of replication of the base pair 6-thioguanine (Gs)/5-methyl-2-pyrimidinone (Th) have been determined by using defined oligomers with the large Klenow fragment of Escherichia coli DNA polymerase I. The insertion efficiency, Vmax/Km (min-1 microM-1), of Th opposite Gs is 1.5 and the insertion efficiency of Gs opposite Th is 0.7. By comparison, the insertion efficiencies of C opposite G and G opposite C are 0.5 and 1.5. The insertion efficiency of the next base, A opposite T, is 2 times greater after the base pair Gs/Th than after G/C. The fidelity of replication with respect to thymine and adenine has misinsertion frequencies, or ratios of the insertion efficiency of the "wrong" base to the "right" base, of 7 x 10(-4) for T opposite Gs (T/Gs), 4 x 10(-6) for T/Th, and a maximum stable misinsertion frequency of 4 x 10(-4) for A/Th. No detectable elongation occurs after an A is inserted opposite a Gs. These values are similar to the misinsertion frequencies of G and C with T and A. The maximum stable misinsertion frequencies with G and C are 4 x 10(-2) for G/Th, 3 x 10(-2) -7 x 10(-3) for Gs/C, and 2.6 x 10(-1) for C/Gs, and the misinsertion frequency is < 1 x 10(-3) for Th/G. The kinetics results and molecular modeling suggest modifications to the Gs/Th base pair that may provide higher levels of fidelity of replication with respect to C and G.

Inactivation of endothelin I by deamidase (lysosomal protective protein).

Deamidase cleaves ester and peptide bonds in various substrates and deamidates protected COOH-terminal amino acids. It preferentially hydrolyzes peptides which contain hydrophobic amino acids in the P1' and/or P1 position. Because the COOH-terminal end of endothelin I contains the hydrophobic sequence -Ile19-Ile20-Trp21-OH, we investigated whether human deamidase, purified from platelets, could inactivate this peptide. We found that deamidase readily cleaved off Trp21 with an acid pH optimum, a Km = 22 microM, a kcat of 1454 min-1, and a kcat/Km of 68 microM-1 min-1. We also found the enzyme to be present in target cells of endothelin, in vascular smooth muscle cells. Extracts of cultured vascular smooth muscle cells cleave both the synthetic fluorescent substrate 5-dimethylaminonaphthalene-1-sulfonyl(Dns)-Phe-Leu-Arg and endothelin I by releasing the COOH-terminal amino acid. The reaction was inhibited by diisopropyl fluorophosphate, benzyloxycarbonyl-Gly-Leu-Phe-CH2Cl, and p-chloromercuribenzenesulfonate, which inhibit the purified deamidase, but not by inhibitors of some other peptidases. The rate of hydrolysis of endothelin I in the soluble, 100,000 x g final supernatant of the homogenized smooth muscle cells was 2.1 mumol/h/mg and 3.1 mumol/h/mg for Dns-Phe-Leu-Arg. Thus, smooth muscles, platelets, and many other tissues which contain the deamidase can inactivate endothelin by cleaving the COOH-terminal tryptophan.

Isolation and characterization of p-coumaroyl esterase from the anaerobic fungus Neocallimastix strain MC-2

An extracellular p-coumaroyl esterase produced by the anaerobic fungus Neocallimastix strain MC-2 released p-coumaroyl groups from 0-[5-0-((E)-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-0-beta -D-xylopyranosyl-(1----4)-D-xylopyranose (PAXX). The esterase was purified 121-fold from culture medium in successive steps involving ultrafiltration column chromatography on S-sepharose and hydroxylapatite, isoelectric focusing, and gel filtration. The native enzyme had an apparent mass of 11 kDa under nondenaturing conditions and a mass of 5.8 kDa under denaturing conditions, suggesting that the enzyme may exist as a dimer. The isoelectric point was 4.7, and the pH optimum was 7.2. The purified esterase had 100 times more activity towards PAXX than towards the analogous feruloyl ester (FAXX). The apparent Km and Vmax of the purified p-coumaroyl esterase for PAXX at pH 7.2 and 40 degrees C were 19.4 microM and 5.1 microM min(-1), respectively. p-Coumaroyl tetrasaccharides isolated from plant cell walls were hydrolyzed at rates similar to that for PAXX, whereas a dimer of PAXX was hydrolyzed at a rate 20-fold lower, yielding 4,4'-dihydroxy-alpha-truxillic acid as an end product. Ethyl and methyl p-coumarates were hydrolyzed at very slow rates, if at all. The purified esterase released p-coumaroyl groups from finely, but not coarsely, ground plant cell walls, and this activity was enhanced by the addition of xylanase and other cell wall-degrading enzymes.

Identification of a highly specific chymase as the major angiotensin II-forming enzyme in the human heart.

Although angiotensin II (Ang II)-forming enzymatic activity in the human left cardiac ventricle is minimally inhibited by angiotensin I (Ang I) converting enzyme inhibitors, over 75% of this activity is inhibited by serine proteinase inhibitors (Urata, H., Healy, B., Stewart, R. W., Bumpus, F. M., and Husain, A. (1990) Circ. Res. 66, 883-890). We now report the identification and characterization of the major Ang II-forming, neutral serine proteinase, from left ventricular tissues of the human heart. A 115,150-fold purification from human cardiac membranes yielded a purified protein with an Mr of 30,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based upon its amino-terminal sequence, the major human cardiac Ang II-forming proteinase appears to be a novel member of the chymase subfamily of chymotrypsin-like serine proteinases. Human heart chymase was completely inhibited by the serine proteinase inhibitors, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, and chymostatin. It was partially inhibited by p-tosyl-L-phenylalanine chloromethyl ketone, but was not inhibited by p-tosyl-L-lysine chloromethyl ketone, and aprotinin. Also, human heart chymase was not inhibited by inhibitors of the other three classes of proteinases. Human heart chymase has a high specificity for the conversion of Ang I to Ang II and the Ang I-carboxyl-terminal dipeptide His-Leu (Km = 60 microM; Kcat = 11,900 min-1; Kcat/Km = 198 min-1 microM-1). Human heart chymase did not degrade several peptide hormones, including Ang II, bradykinin, and vasoactive intestinal peptide, nor did it form Ang II from angiotensinogen. The high substrate specificity of human heart chymase for Ang I distinguishes it from other Ang II-forming enzymes including Ang I converting enzyme, tonin, kallikrein, cathepsin G, and other known chymases.

Interaction of plasminogen and fibrin in plasminogen activation.

Glu1-, Lys77-, miniplasminogens, kringle 1-3, kringle 1-5A, and kringle 1-5R were able to bind with fibrin, while microplasminogen and kringle 4 did not bind significantly. Kringle 1-5A, but not kringle 1-3, effectively inhibited the binding of Glu1-, Lys77-, and miniplasminogens with fibrin. Miniplasminogen also inhibited the binding of Glu1-plasminogen with fibrin. The binding of kringle 1-3 with fibrin was blocked by mini- or Glu1-plasminogen. It is therefore evident that there are two fibrin-binding domains in plasminogen and that the one in kringle 5 is of higher affinity than that in kringle 1-3. CNBr cleavage products of fibrinogen effectively enhanced the activation of Glu1-, Lys77-, or miniplasminogens, but not microplasminogen, by tissue-type plasminogen activator. Kringle 1-5, but not kringle 1-3, dose-dependently inhibited the enhancement by fibrinogen degradation products of Glu1-plasminogen activation by the activator. Lysine and epsilon-aminocaproic acid could inhibit the binding of plasminogens and plasminogen derivatives with fibrin and block the enhancement effect of fibrinogen degradation products on plasminogen activation. The data clearly illustrate that the binding of plasminogen with fibrin, mainly determined by kringle 5, is essential for effective activation by tissue-type plasminogen activator. However, the presence of kringle 1-4 in the plasminogen molecule is required for the full enhancing effect since the kcat/Km of miniplasminogen activation in the presence of fibrinogen degradation products was 8.2 microM-1 min-1 which is significantly less than 52.0 microM-1 min-1 of Glu1-plasminogen.

Localization and characterization of 35S-t- butylbicyclophosphorothionate binding in rat brain: an autoradiographic study

35S-t-butylbicyclophosphorothionate (TBPS) binding to slide-mounted rat brain sections was characterized for subsequent autoradiographic analysis. Cortical brain mash slices, preincubated with EDTA to remove endogenous GABA, were used for biochemical characterization. Steady state for 35S-TBPS binding was reached by 3 hr of incubation at 22 degrees C. The association rate constant (K1) and dissociation rate constant (K2) were 0.377 min-1 microM-1 and 0.011 min-1, respectively. Dissociation was monophasic and slow (t1/2 = 80 min). The kinetically derived KD was 29.4 nM. Scatchard analysis indicated a single population of binding sites with a KD of 21.0 +/- 2.2 nM and a Bmax of 1.59 +/- 0.13 pmol/mg protein. Both picrotoxin and muscimol inhibited 35S-TBPS binding completely with IC50s of 251 +/- 13 nM and 203 +/- 41 nM and nHs of 0.98 and 1.4, respectively. The distribution of 35S-TBPS binding sites in the rat brain resembles that of other ligands that bind to GABAA receptor complex with some regionally specific differences. Regions with a high degree of 35S-TBPS binding included the inferior colliculus, medial septal nucleus, central and paracentral nuclei of the thalamus, olfactory tubercle, zona incerta, dentate gyrus, and substantia nigra. 35S-TBPS preferentially bound to the molecular vs granular layer of the cerebellum. Omission of the preincubation markedly but variably decreased 35S-TBPS binding. The greatest regional decreases occurred in areas with a high degree of GABA synthesis. In addition, 35S-TBPS binding was inhibited to different degrees in the cell layers of the cerebellum. The addition of 1 microM GABA to the incubation medium of preincubated slices also produced variable decreases in 35S-TBPS binding to cerebellar layers. These findings support previous studies that demonstrate GABAA receptor heterogeneity. Our study confirms the suitability of 35S-TBPS for use as a ligand in autoradiography and demonstrates that the distribution of 35S-TBPS binding sites is significantly influenced by the preincubation-incubation conditions used.

Surface patterning of GaAs by CCl 2F 2 reactive ion etching

Reactive ion etching of GaAs in a CCl 2F 2 plasma has been studied for a range of powers at pressures of less than 5.3 Pa. The etching behaviour has been characterised in terms of anisotropy, etch rate and surface roughness. The influence of electrode diameter and coverplate materials has also been examined. These studies have revealed two regimes of etching behaviour, a fact not previously reported at these pressures. At high power densities (0.22–0.44 W cm −2) and low pressures (1.3–2.7 Pa), the GaAs etch rate was relatively low, and the cleanliness of the etched surface was consistently high. Photoresist was readily etched in this region. At lower power densities and higher pressures, GaAs was etched significantly faster (up to 0.25 microm min −1 whilst photoresist was eroded very slowly. Etching in this regime was much more likely to produce unwanted surface features, which are ascribed to the presence of initial wafer contamination. An in situ plasma clean has been demonstrated as a means for overcoming this latter problem.

Activation and cleavage of the carbon-cobalt bond of adeninylethylcobalamin by diol dehydrase.

Adeninylethylcobalamin (AdeEtCbl) underwent cleavage of the C-Co bond by interaction with apoprotein of diol dehydrase from Klebsiella pneumoniae ATCC 8724, although this analog was quite inactive as coenzyme. Spectroscopic observation indicates that AdeEtCbl was converted to the enzyme-bound hydroxocobalamin without intermediates. The conversion was stoichiometric (1:1) and obeyed the second-order reaction kinetics (k = 0.027 min-1 microM-1 at 37 degrees C) depending upon concentrations of apoprotein and AdeEtCbl. This suggests that the complex formation is the rate-determining step and that AdeEtCbl undergoes rapid C-Co bond cleavage once it binds to the apoenzyme. Substrates and oxygen did apparently not affect the rate of the C-Co bond cleavage. The experiments using [adenine-U-14C]AdeEtCbl and [1(3)-3H]glycerol demonstrated that 9-ethyladenine was the only product formed from the adeninylethyl group of AdeEtCbl during the conversion and that an additional hydrogen atom in the 9-ethyladenine is not derived from the substrate. 1H NMR measurement of the 9-ethyladenine formed enzymatically from AdeEtCbl and DL-1,2-[1,1,2-2H3]propanediol also led to the same conclusion. All of these results indicate that the C-Co bond of AdeEtCbl is activated by diol dehydrase and undergoes heterolysis forming Co(III) and a carbanion or a carbanion-like species, in clear contrast to the homolysis of the C-Co bond of adenosylcobalamin in the normal catalytic process. 9-Ethyladenine formed remained tightly associated with the enzyme. Longer chain homologs, i.e. adeninylpropylcobalamin, adeninylbutylcobalamin, and adeninylpentylcobalamin did not undergo such cleavage of the C-Co bond by diol dehydrase.